Rat Renal Tubular Cell Cultures Research Articles

Ectopic overexpression of haem oxygenase‐1 protects kidneys from carboplatin‐mediated apoptosis

We previously reported that the activation of the nuclear factor of activated T-lymphocyte-3 (NFAT3) by carboplatin leads to renal apoptosis as a result of oxidative stress, which is reversed by N-acetylcysteine. Herein, we extend our previous work to provide evidence of the molecular mechanisms of haem oxygenase (HO)-1 in protecting against injury. Protective mechanisms of HO-1 in carboplatin-mediated renal apoptosis were examined in C57BL/6 mice and rat renal tubular cells (RTC) with HO-1 induction or inactivation/knockdown. The HO-1, induced by cobalt protoporphyrin, protected against carboplatin-induced renal injury in vivo. This protection was decreased by an inhibitor of HO-1 action, tin protoporphyrin. In cultures of RTC, carboplatin-induced apoptosis was similarly affected by HO-1 overexpression or knockdown. Carboplatin-mediated NFAT3 activation and apoptosis involve activation of the signalling kinases, extracellular signal regulated kinase, Jun N-terminal kinase and protein kinase C, and such activation was reversed in cells overexpressing HO-1. Both products of the HO-1 reaction, CO and bilirubin, inhibited (by 30-40%) NFAT3 activation and production of the pro-apoptotic proteins Bcl-XS/Bax. Additionally, the activation of NFκB was markedly decreased by HO-1 induction. HO-1 and its reaction products show anti-apoptotic effects in carboplatin-mediated renal injury. A novel functional NFAT3 binding site identified in the rat HO-1 promoter region was involved in producing a 1.5-fold to 2.5-fold increase in HO-1 induction by carboplatin. Nevertheless, only HO-1 overexpression and activation prior to the carboplatin challenge provided protection against carboplatin-induced injury.

Effect of aroclor 1248 and two pure PCB congeners on phospholipase D activity in rat renal tubular cell cultures

This paper elucidates the effect of different polychlorinated biphenyls (PCBs) on the phospholipase D (PLD) activity in soluble and particulate fractions of rat renal proximal tubular culture cells. Treatment with Aroclor 1248 (a commercial PCB mixture) caused a marked increase in the activity of PLD in intact renal tubular cells. The PLD activity was increased by Aroclor 1248 in the particulate fraction while the enzyme activity was unaffected in the soluble fraction. This work also shows that PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) can increase the activity of PLD only in the particulate fraction. The exposure of cell cultures to PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener) does not alter PLD activity. These results suggest that PCB effects are structure dependent. Therefore, in order to clarify the molecular mechanism of activation of PLD by PCBs, the contents of immunoreactive PLD were examined by immunoblot analysis. Renal tubular cells expressed a PLD protein of 120 kDa corresponding with the PLD1 mammalian isoform in both the particulate and the soluble fraction. Aroclor 1248, PCB 153, and PCB 77 do not induce changes in the levels of PLD protein. These data indicate that PCBs, particularly nonplanar congeners, increase PLD activity. Moreover, these changes could not be demonstrated in the enzyme content in rat renal tubular cell cultures.

Differential effects of PCBs on the induction of apoptosis machinery and PKCα translocation in rat renal tubular cell cultures

We have demonstrated previously [Pérez-Reyes, P.L., Sánchez-Alonso, J.A., López-Aparicio, P., Recio, M.N., Pérez-Albarsanz, M.A., 2001. Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures. Biosci. Rep. 6, 765–778] that the polychlorinated biphenyls (PCBs) cause loss of cell viability and accelerate apoptosis in cell kidney cultures. Further investigations are necessary to elucidate the mechanism of apoptosis induction. In this way, we have analyzed in the present work the effects of PCBs on protein kinase C (PKC, a protein family intimately involved in the regulation of cell survival) and the expression of two proapoptotic (caspase-3 and Bax) and one antiapoptotic (Bcl-2) proteins. Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), PCB 153 (2,2′,4,4′,5,5′-hexachlorobiphenyl, a di- ortho-substituted nonplanar congener) and PCB 77 (3,3′,4,4′-tetrachlorobiphenyl, a non- ortho-substituted planar congener), significantly increased PKCα activity compared to control cells in the cytosolic and particulate cell fractions, and increased the PKCα protein content in the particulate fraction. The nonplanar PCB 153 showed stronger effects than the coplanar congener PCB 77. In addition, Aroclor 1248 decreased both, procaspase-3 levels and the Bcl-2/Bax protein ratio. These findings indicate that PCBs, particularly nonplanar congeners, can induce apoptosis in primary renal tubular cells through the PKCα, caspase-3 and Bcl-2/Bax pathway.

Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures.

The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis. Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner. The two PCB congeners assessed can also induce apoptosis. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3',4,4-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2',4,4',5,5'-hexachlorobiphenyl). This correlated with the loss of cell viability since the planar compound is much more cytotoxic. The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.

Redistribution of cPLA 2 in rat renal tubular cell cultures in response to PCBs

The influence of different polychlorinated biphenyls (PCBs) upon the cytosolic phospholipase A 2 (cPLA 2) redistribution to the particulate fraction has been investigated in rat renal proximal tubule culture cells. Treatment with Aroclor 1248 increased PLA 2 activity in the particulate fraction in a concentration-dependent manner using two radioactive substrates. However, the activity of PLA 2 in the cytosolic fraction decreased. This work also shows that 2,2′,4,4′,5,5′-hexachlorobiphenyl (HCB) (a di- ortho-substituted nonplanar congener) can increase the activity of PLA 2 in the particulate fraction and decrease the enzyme activity in the cytosolic fraction. The exposure of cell cultures to 3,3′,4,4′-tetrachlorobiphenyl (TCB) (a non- ortho-subtituted planar congener) does not alter PLA 2 activity. These results suggest that PCBs, depending on their planar or nonplanar structures, cause a translocation of the enzyme from the cytosol to membranes. To evaluate this possibility, the contents of immunoreactive cPLA 2 were examined by immunoblot analysis in the high-speed supernatant and the particulate fraction of treated cell cultures. The increases/decreases in the amounts of cPLA 2 protein agree with the increases/decreases of PLA 2 activity previously cited. These data demonstrate that the PCB-stimulated redistribution of cPLA 2 to membranes is associated, at least in part, with the changes detected in the activity of the enzyme.

Selective fatty acid release from intracellular phospholipids caused by PCBs in rat renal tubular cell cultures

The purpose of this study was to explore the influence of different polychlorinated biphenyls (PCBs) upon the release of oleic and palmitic acid from the intracellular lipids, which were previously labeled with [ 3H]oleic or [ 3H]palmitic acid, respectively. Studies have been realized with Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), and two pure PCB congeners: 3,3′,4,4′-tetrachlorobiphenyl (a non-ortho-substituted planar congener) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (a di-ortho-substituted nonplanar congener). The treatment of cells with Aroclor 1248 increased [ 3H]oleic acid release in a concentration-dependent manner. Our results showed that only the di-ortho-substituted congener which prefers a nonplanar configuration stimulated the release of [ 3H]oleic acid from the intracellular phospholipids to the culture medium, while the exposure of cell cultures to the chosen non-ortho-substituted coplanar congener did not alter the release of [ 3H]oleic acid to the culture medium. Finally, none of the PCBs studied could increase the release of [ 3H]palmitic acid from the intracellular stores significantly. The possibility that these differential alterations in the fatty acid release affect cell function during PCB exposure should therefore be postulated.

Effect of Aroclor 1248 and Two Pure PCB Congeners upon the Release of Arachidonic Acid from the Intracellular Phospholipids of Rat Renal Tubular Cell Cultures

The exposure of rat tubular cell cultures to different polychlorinated biphenyls (PCBs) stimulated the release of arachidonic acid from the intracellular phospholipids, which were previously labeled with [3H]ara chidonic acid. Studies have been realized with Aroclor 1248 (a commercial PCBs mixture with 48% chlorine by weight) and two pure PCB congeners: 3,3′,4,4′-tetrachlorobyphenyl (a coplanar compound) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (adiortho-substituted nonplanar congener). When Aroclor 1248 was added, a relation between Aroclor concentration and arachidonate release to the culture medium was found. This work concludes that only thediortho-substituted congener that prefers a nonplanar configuration can increase the release of [3H]arachidonic acid from the intracellular stores to the culture medium, while the exposure of cell cultures to the chosen coplanar congener does not alter the release of [3H]arachidonic acid to the culture medium in a significant manner. This means that the toxic effect of PCBs could be due, at least in part, to the coplanarity or noncoplanarity of these compounds.

Effect of Aroclor 1248 and Two Pure PCB Congeners upon the Membrane Fluidity of Rat Renal Tubular Cell Cultures

The influence of different polychlorinated biphenyls (PCBs) upon dynamic properties of membranes from rat renal tubular cell cultures has been investigated. Studies have been realized with Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), and two pure PCB congeners: 3,3′,4,4′-tetrachlorobyphenyl (a coplanar compound) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (anortho-substituted nonplanar congener). Changes in the membrane fluidity were measured by the fluorescence polarization technique using the probe diphenylhexatriene. The treatment of cells with Aroclor 1248 increased membrane fluidity in a dose-dependent manner. Even though rat renal tubular cell cultures accumulated only a low amount of PCBs when cells were incubated in presence of the toxicant, that small quantity was enough to modify the membrane fluidity. Interestingly, a significant increase of membrane fluidity was found in presence of 2,2′,4,4′,5,5′-hexachlorobiphenyl, but no significant differences were detected in the presence of 3,3′,4,4′-tetrachlorobiphenyl. This means that changes in the membrane fluidity induced by the different PCB congeners are related to the coplanarity or lack of coplanarity of the toxic compounds.